Compositions and methods for glioblastoma treatment

ABSTRACT

The present disclosure concerns an oncolytic virus for the treatment of cancer, such as in brain cancer, for example glioblastoma. The oncolytic virus may exhibit reduced levels of neurotoxicity. The oncolytic virus may be an isolated viral particle capable of producing a cDNA polynucleotide that includes a sequence according to SEQ ID NO: 1 when the virus is in a host cell. The oncolytic virus may be an isolated viral particle that includes an RNA polynucleotide that includes a sequence according to SEQ ID NO: 2. The oncolytic virus may be an isolated viral particle having a genome that includes open reading frames that encode: proteins having sequences comprising SEQ ID NOs: 3, 4, 5, 6 and 7; or variants thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/020,490 filed on Sep. 14, 2020, which is a continuation of U.S.patent application Ser. No. 15/155,983 filed May 16, 2016 (now U.S. Pat.No. 10,772,951), which is a continuation of U.S. patent application Ser.No. 14/123,057 filed Nov. 27, 2013 (now U.S. Pat. No. 9,364,532), whichis a national phase entry of PCT/CA2012/050385 filed on Jun. 7, 2012,which claims priority to U.S. Provisional Patent Application No.61/494,628 filed Jun. 8, 2011, which are all incorporated herein byreference.

FIELD

The present disclosure relates to rhabdoviruses and their use as anoncolytic treatment. More specifically, the present disclosure relatesto Farmington rhabdovirus and its use in the treatment of glioblastoma.

BACKGROUND

This section provides background information related to the presentdisclosure which is not necessarily prior art.

Brain cancer is the leading cause of cancer-related death in patientsyounger than age 35 and accounts for roughly 10% of all cancersdiagnosed in North America. Treatment of brain tumours is complicated bythe fact that there are more than 120 different types, which range fromlow grade astrocytomas to high grade glioblastomas (GBM). Malignantgliomas, such as GBM, are by far the most common brain cancer found inadults and one of the most difficult to treat. Even with aggressivesingle and multimodal treatment options such as surgery, chemotherapy,radiation and small molecule inhibitors, the survival has remainedunchanged over the past three decades with a median survival of lessthan one year after diagnosis. Reasons for the failure of conventionaltreatments is multifactorial including the highly infiltrative/invasivenature of GBM, limitation of drug delivery through the blood brainbarrier and neural parenchyma, and genetic heterogeneity resulting inintrinsic resistance to available treatments and the rise of aggressiveresistant clones. Therefore, there is a dire requirement for newtreatment options, which has led to the renaissance of oncolytic viraltherapy for GBM.

Currently, the efficacy and safety of several oncolytic viruses withvarious tumour targeting strategies are being evaluated in the lab andclinic against GBM. The rhabdovirus vesicular stomatitis virus (VSV)constitutes one of these efficacious viruses being tested preclinically.However, a desired route of viral administration for GBM isintracerebral delivery, which is not currently possible with VSV due toits neurotoxicity.

SUMMARY

It is an object of the present disclosure to obviate or mitigate atleast one disadvantage of previous oncolytic viruses. For example, theoncolytic virus of the present disclosure may exhibit reduced levels ofneurotoxicity.

The present technology includes systems, methods, processes, articles,and compositions that relate to rhabdoviruses, such as Farmingtonrhabdovirus, and related nucleotide and protein sequences thereof, andthe use of such in oncolytic treatments, for example treatments forglioblastoma.

According to one aspect of the present disclosure, there is provided anisolated viral particle capable of producing a cDNA polynucleotide thatincludes a sequence according to SEQ ID NO: 1 when the virus is in ahost cell.

According to another aspect of the present disclosure, there is providedan isolated viral particle that includes an RNA polynucleotide thatincludes a sequence according to SEQ ID NO: 2.

According to still another aspect of the present disclosure, there isprovided an isolated viral particle having a genome that includes openreading frames that encode: a protein having a sequence comprising SEQID NO: 3, or a variant thereof; a protein having a sequence comprisingSEQ ID NO: 4, or a variant thereof; a protein having a sequencecomprising SEQ ID NO: 5, or a variant thereof; a protein having asequence comprising SEQ ID NO: 6, or a variant thereof; and a proteinhaving a sequence comprising SEQ ID NO: 7, or a variant thereof.

The variant of a reference protein may be a protein that has a sequencewhich is at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to the sequence of the reference protein, and wherethe variant protein maintains the same biological function as thereference protein.

In some examples, at least one of the open reading frames encodes aprotein having a sequence which is a sequence selected from the groupconsisting of SEQ ID NOs: 3, 4, 5, 6, and 7. In such examples, thevariant of a reference protein may be a protein having a sequence whichis at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%identical to the sequence of the reference protein, and where thevariant protein maintains the same biological function as the referenceprotein.

The viral genome may include open reading frames that encode: a proteinhaving a sequence comprising SEQ ID NO: 3; a protein having a sequencecomprising SEQ ID NO: 4; a protein having a sequence comprising SEQ IDNO: 5; a protein having a sequence comprising SEQ ID NO: 6; and aprotein having a sequence comprising SEQ ID NO: 7.

The isolated viral particle may further include at least one additionalopen reading frame for encoding at least one additional protein. Theadditional protein may be an immunogenic protein.

According to another aspect of the present disclosure, there is providedan isolated viral particle capable of producing a cDNA polynucleotidewhen the virus is in a host cell, the cDNA polynucleotide having asequence that includes: SEQ ID NO: 8, or a conservative variant thereof;SEQ ID NO: 9, or a conservative variant thereof; SEQ ID NO: 10, or aconservative variant thereof; SEQ ID NO: 11, or a conservative variantthereof; SEQ ID NO: 12, or a conservative variant thereof; and promotersthereof.

The conservative variant of a sequence of nucleotides may be a sequencethat is at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to the reference sequence of nucleotides. Theconservative variant may be a sequence comprising one or more silentsubstitutions.

According to still another aspect of the present disclosure, an isolatedviral particle according to the present disclosure may be used for thetreatment of cancer. The cancer may be a brain cancer. The brain cancermay be a glioblastoma. The isolated viral particle may be used to infecta cell where the infected cell is for the treatment of cancer.

According to still another aspect of the present disclosure, an isolatedviral particle according to the present disclosure may be used forinducing an immunogenic response in a person administered the virus. Theimmunogenic response may be an anti-cancer response. The isolated viralparticle may be used to infect a cell where the infected cell is use togenerate the immunogenic response.

The isolated viral particle may be formulated for direct delivery to thecentral nervous system, outside the blood/brain barrier, inside theblood/brain barrier, or any combination thereof. The isolated viralparticle may be formulated for administration via intrathecal,intravenous or intracranial injection.

The infected cell may be formulated for direct delivery to the centralnervous system, outside the blood/brain barrier, inside the blood/brainbarrier, or any combination thereof. The infected cell may be formulatedfor administration via intrathecal, intravenous or intracranialinjection.

According to still another aspect of the present disclosure, there isprovided a method for treating cancer comprising administering anisolated viral particle according to the present disclosure to a patienthaving cancer. The cancer may be a brain cancer. The brain cancer may bea glioblastoma.

The isolated viral particle may be administered to the patient directly.For example, the isolated viral particle may be administered directly tothe central nervous system, outside the blood/brain barrier, inside theblood/brain barrier, or any combination thereof. The isolated viralparticle may be administered to the patient intrathecally, intravenouslyor via intracranial injection.

Alternatively, a cell may be infected with the isolated viral particleand the infected cell may be administered to the patient. The infectedcell may be administered directly to the central nervous system, outsidethe blood/brain barrier, inside the blood/brain barrier, or anycombination thereof. The infected cell may be administered to thepatient intrathecally, intravenously or via intracranial injection.

According to still another aspect of the present disclosure, there isprovided a method for inducing an immunogenic response in a patient, themethod including administering an isolated viral particle according tothe present disclosure to the patient.

The isolated viral particle may be administered to the patient directly.For example, the isolated viral particle may be administered directly tothe central nervous system, outside the blood/brain barrier, inside theblood/brain barrier, or any combination thereof. The isolated viralparticle may be administered to the patient intrathecally, intravenouslyor via intracranial injection.

Alternatively, a cell may be infected with the isolated viral particleand the infected cell may be administered to the patient. The infectedcell may be administered directly to the central nervous system, outsidethe blood/brain barrier, inside the blood/brain barrier, or anycombination thereof. The infected cell may be administered to thepatient intrathecally, intravenously or via intracranial injection.

According to a yet further aspect of the present disclosure, there isprovided a kit for the treatment of cancer in a patient, the kitincluding: the isolated viral particle according to any one of claims 1to 12; and instructions for administration of the isolated viralparticle to the patient.

The cancer may be a brain cancer. The brain cancer may be aglioblastoma.

The isolated viral particle may be formulated for direct delivery to thecentral nervous system, outside the blood/brain barrier, inside theblood/brain barrier, or any combination thereof. The isolated viralparticle may be formulated for administration via intrathecal,intravenous or intracranial injection.

Alternatively, the isolated viral particle may be formulated forinfection of a cell where the cell is for delivery to the centralnervous system, outside the blood/brain barrier, inside the blood/brainbarrier, or any combination thereof. The cell may be for administrationvia intrathecal, intravenous or intracranial injection.

Other aspects and features of the present disclosure will becomeapparent to those ordinarily skilled in the art upon review of thefollowing description of specific embodiments in conjunction with theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described, by way ofexample only, with reference to the attached Figures.

FIG. 1A is a map showing the region FMT virus was isolated; FIG. 1B is aAmino Acid Blast alignment of 5 FMT putative open reading frames (ORFs)(N, P, G, M and L) reveals little sequence homology to known sequencesin the NCBI database; FIG. 1C is a SDS PAGE gel of FMT virus showing 4of the 5 predicted FMT ORF proteins; FIG. 1D is an illustration of thePhylogenic tree of various rhabdoviruses; FIG. 1E is an Example of aFully replicative GFP expressing FMT strain; and FIG. 1F is an Electronmicrogram of FMT virion (adapted from Tesh et al. Emerging Infect. Dis.2002).

FIG. 2A illustrates an intracranial neurotoxicity screen forrhabdoviruses;

FIG. 2B is a graph illustrating motor function assessment by time onrotorod after intracerebral injection of FMT in Balb/C mice; FIG. 2C isa graph illustrating a FMT MTD determination in Balb/C mice injected IVwith increasing doses (3×e6 pfu-3e9 pfu) of FMT virus; FIG. 2D is atable illustrating detection of viable FMT virus 3 monthspost-inoculation; FIG. 2E shows histopathology photographs of Brainfollowing intracerebral inoculation with FMT, VSV or PBS.

FIG. 3A is a table showing a summary of FMT virus cytotoxicity in vitro;

FIG. 3B is a set of graphs illustrating that FMT is a potent andselective killer of GBM cell lines; and FIG. 3C is a table showing anassessment of FMT potency against tumour and normal cells.

FIG. 4A is a set of photographs of a U-87 MG Human Glioma XenograftModel; FIG. 4B is a graph illustrating a Kaplan Meier survival plot ofanimals treated with a single dose IC (1e5 pfu) or IV (5e8 pfu); FIG. 4Cis a fluorescence micrograph of mock infected mouse brain (with GFPtagged U87MG tumour) versus FMT treated mouse brain;

FIG. 4D is a graph illustrating a Late Syngeneic mouse GBM model usingthe CT2A cell line treated both IC and IV with FMT or PBS.

FIG. 5A is a photograph showing C57/B6 mice implanted with CT2A murineglioma cells into the striatum and treated with FMT and cured of initialtumour then challenged with CT2A cells implanted into the striatumversus Naive mice implanted with CT2A cells as a control; FIG. 5B is agraph illustrating C57/B6 mice implanted with CT2A murine glioma cellsinto the striatum and allowed to grow tumours received either anti-CD8polyclonal serum injections to remove CD8+ T cells or matched pre-immuneserum as a control then both groups were treated with a singleintracranial dose of FMT to induce tumour regressions.

FIG. 6A is a photograph of a Western blot of human brain tumour cells(SNB19) and primary normal human astrocytes (NHA) showing FMT virusinfection and protein production; FIG. 6B is a graph illustrating thegrowth curve of FMT virus production in SNB19 and NHA cells; FIG. 6Cshows fluorescence microscopy photographs of GFP-expressing rec-FMT orMaraba-Δ51 virus added to a GM38 cell monolayer with and withouttreatment with the type I interferon inhibitor B18R protein derived fromvaccinnia virus (VV-B18R); FIG. 6D is a graph illustrating plaque andinfectious foci size measured from fluorescence microscopy; FIG. 6E is agraph illustrating an interferon bioassay in PC3 cells.

FIG. 7A is a graph illustrating viral titer determined inTeratocarcinoma and differentiated NT2 cells infected with the indicatedviruses; FIG. 7B is a graph illustrating teratocarcinoma anddifferentiated NT2 cells infected with the indicated viruses and assayedfor viability using Alamar blue metabolic dye; FIG. 7C shows photographsof Western blots of several components of the cellular apoptoticsignaling cascade following infection of either tumour (SNB19 or normalcells (NHA); FIG. 7D is an illustration of a schematic of cellularapoptosis signaling cascade.

DESCRIPTION Definitions

Throughout the present disclosure, several terms are employed that aredefined in the following paragraphs.

As used herein, the words “desire” or “desirable” refer to embodimentsof the technology that afford certain benefits, under certaincircumstances. However, other embodiments may also be desirable, underthe same or other circumstances. Furthermore, the recitation of one ormore desired embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the technology.

As used herein, the word “include,” and its variants, is intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this technology. Similarly, theterms “can” and “may” and their variants are intended to benon-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present technology that do not contain those elements orfeatures.

Although the open-ended term “comprising,” as a synonym ofnon-restrictive terms such as including, containing, or having, is usedherein to describe and claim embodiments of the present technology,embodiments may alternatively be described using more limiting termssuch as “consisting of” or “consisting essentially of.” Thus, for anygiven embodiment reciting materials, components or process steps, thepresent technology also specifically includes embodiments consisting of,or consisting essentially of, such materials, components or processesexcluding additional materials, components or processes (for consistingof) and excluding additional materials, components or processesaffecting the significant properties of the embodiment (for consistingessentially of), even though such additional materials, components orprocesses are not explicitly recited in this application. For example,recitation of a composition or process reciting elements A, B and Cspecifically envisions embodiments consisting of, and consistingessentially of, A, B and C, excluding an element D that may be recitedin the art, even though element D is not explicitly described as beingexcluded herein.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. Disclosures of rangesare, unless specified otherwise, inclusive of endpoints and include alldistinct values and further divided ranges within the entire range.Thus, for example, a range of “from A to B” or “from about A to about B”is inclusive of A and of B. Disclosure of values and ranges of valuesfor specific parameters (such as temperatures, molecular weights, weightpercentages, etc.) are not exclusive of other values and ranges ofvalues useful herein. It is envisioned that two or more specificexemplified values for a given parameter may define endpoints for arange of values that may be claimed for the parameter. For example, ifParameter X is exemplified herein to have value A and also exemplifiedto have value Z, it is envisioned that Parameter X may have a range ofvalues from about A to about Z. Similarly, it is envisioned thatdisclosure of two or more ranges of values for a parameter (whether suchranges are nested, overlapping or distinct) subsume all possiblecombination of ranges for the value that might be claimed usingendpoints of the disclosed ranges. For example, if Parameter X isexemplified herein to have values in the range of 1-10, or 2-9, or 3-8,it is also envisioned that Parameter X may have other ranges of valuesincluding 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9.

“A” and “an” as used herein indicate “at least one” of the item ispresent; a plurality of such items may be present, when possible.

“About” when applied to values indicates that the calculation or themeasurement allows some slight imprecision in the value (with someapproach to exactness in the value; approximately or reasonably close tothe value; nearly). If, for some reason, the imprecision provided by“about” is not otherwise understood in the art with this ordinarymeaning, then “about” as used herein indicates at least variations thatmay arise from ordinary methods of measuring or using such parameters.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, a virus that has “reduced levels of neurotoxicity” or“reduced neurotoxicity” would be understood to refer to a virus that,when injected into the right striatum of a mouse brain at a given dose,results in a mouse with fewer signs of neurotoxicity (for example, weighloss, piloerection, hind leg paralysis, morbidity and mortality) than amouse which is injected with wild-type maraba virus.

As used herein, “substantially no level of neurotoxicity” or“substantially no neurotoxicity” would be understood to refer to a virusthat, when injected intracerebrally into a mouse at 1e6 pfu, results ina mouse with no detectable signs of reduced motor function as measuredby time on a rotorod compared to the mouse before injection with thevirus.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Of the more than 250 currently identified rhabdoviruses, severalisolated wild type rhabdoviruses were determined to be effective atkilling CNS tumour cell lines while retaining attenuation in normalhuman astrocytes and post-mitotic neurons. Several of these potent viralisolates were also determined to demonstrate remarkable attenuation,resulting in 100% survival after intracerebral inoculation. This is instriking contrast to previously tested Maraba and VSV viruses.

Generally, the present disclosure provides an oncolytic virus for thetreatment of cancer. Oncolytic viruses may be used to treat cancer bydirectly administering the virus to a patient, or by infecting a cellwith the virus and administering the infected cell to the patient todeliver the virus. The cell to be infected by the virus may be a cancercell from the patient. In some examples, the cancer to be treated isbrain cancer, such as malignant glioma. One example of a malignantglioma is glioblastoma. The oncolytic virus may exhibit reduced levelsof neurotoxicity.

The oncolytic virus may be an isolated viral particle capable ofproducing a cDNA polynucleotide that includes a sequence according toSEQ ID NO: 1 when the virus is in a host cell.

The oncolytic virus may be an isolated viral particle that includes anRNA polynucleotide that includes a sequence according to SEQ ID NO: 2.

The oncolytic virus may be an isolated viral particle having a genomethat includes open reading frames that encode: a protein having asequence comprising SEQ ID NO: 3, or a variant thereof; a protein havinga sequence comprising SEQ ID NO: 4, or a variant thereof; a proteinhaving a sequence comprising SEQ ID NO: 5, or a variant thereof; aprotein having a sequence comprising SEQ ID NO: 6, or a variant thereof;and a protein having a sequence comprising SEQ ID NO: 7, or a variantthereof.

The variant of a reference protein may be a protein that has a sequencewhich is at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to the sequence of the reference protein, and wherethe variant protein maintains the same biological function as thereference protein.

In some examples, at least one of the open reading frames encodes aprotein having a sequence which is a sequence selected from the groupconsisting of SEQ ID NOs: 3, 4, 5, 6, and 7. In such examples, thevariant of a reference protein may be a protein having a sequence whichis at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%identical to the sequence of the reference protein, and where thevariant protein maintains the same biological function as the referenceprotein.

The viral genome may include open reading frames that encode: a proteinhaving a sequence comprising SEQ ID NO: 3; a protein having a sequencecomprising SEQ ID NO: 4; a protein having a sequence comprising SEQ IDNO: 5; a protein having a sequence comprising SEQ ID NO: 6; and aprotein having a sequence comprising SEQ ID NO: 7.

The isolated viral particle may further include at least one additionalopen reading frame for encoding at least one additional protein. Theadditional protein may be an immunogenic protein.

The oncolytic virus may be an isolated viral particle capable ofproducing a cDNA polynucleotide when the virus is in a host cell, thecDNA polynucleotide having a sequence that includes: SEQ ID NO: 8, or aconservative variant thereof; SEQ ID NO: 9, or a conservative variantthereof; 2894-3340 of SEQ ID NO: 10, or a conservative variant thereof;SEQ ID NO: 11, or a conservative variant thereof; SEQ ID NO: 12, or aconservative variant thereof; and promoters thereof.

The conservative variant of a sequence of nucleotides may be a sequencethat is at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to the reference sequence of nucleotides. Theconservative variant may be a sequence comprising one or more silentsubstitutions.

An isolated viral particle according to the present disclosure may beused for the treatment of cancer. The cancer may be a brain cancer. Thebrain cancer may be a glioblastoma. The isolated viral particle may beused for the treatment of cancer by infecting a cell with the virus andthe infected cell may be used to deliver the virus to a patient.Techniques for infecting a cell with a virus and using the infected cellto deliver the virus are discussed in, for example: Power A T, et al.Carrier cell-based delivery of an oncolytic virus circumvents antiviralimmunity. Mol Ther. 2007 Jan.; 15(1):123-30; and Tyler M A, et al.Neural stem cells target intracranial glioma to deliver an oncolyticadenovirus in vivo. Gene Ther. 2009 Feb.; 16(2):262-78.

An isolated viral particle according to the present disclosure may beused to induce an immunogenic response in a person administered thevirus. The immunogenic response may be an anti-cancer response. Theisolated viral particle may be used induce an immunogenic response byinfecting a cell with the virus and the infected cell may be used todeliver the virus to the person.

The isolated viral particle may be formulated for direct delivery to thecentral nervous system, outside the blood/brain barrier, inside theblood/brain barrier, or any combination thereof. For example theisolated viral particle may be formulated for administration viaintrathecal, intravenous or intracranial injection.

An isolated viral particle according to the present disclosure may beused in a a method for treating cancer, where the method includesadministering an isolated viral particle according the presentdisclosure to a patient having cancer. The cancer may be a brain cancer.The brain cancer may be a glioblastoma. The isolated viral particle maybe administered to the patient directly to the central nervous system,outside the blood/brain barrier, inside the blood/brain barrier, or anycombination thereof. For example, the isolated viral particle may beadministered intrathecally, intravenously or via intracranial injection.The isolated viral particle may be directly administered to the patientor may be administered to the patient by infecting a cell with the virusand administering the infected cell to the patient.

An isolated viral particle according to the present disclosure may beused in a method for inducing an immunogenic response in a patient,where the method includes administering an isolated viral particleaccording to the present disclosure to the patient. The isolated viralparticle may be administered to the patient directly to the centralnervous system, outside the blood/brain barrier, inside the blood/brainbarrier, or any combination thereof. For example, the isolated viralparticle may be administered intrathecally, intravenously or viaintracranial injection.

An isolated viral particle according to the present disclosure may beincluded in a kit for the treatment of cancer in a patient, the kitincluding: the isolated viral particle according to the presentdisclosure; and instructions for administration of the isolated viralparticle to the patient. The cancer may be a brain cancer. The braincancer may be a glioblastoma. The isolated viral particle may beformulated for direct delivery to the central nervous system, outsidethe blood/brain barrier, inside the blood/brain barrier, or anycombination thereof. For example the isolated viral particle may beformulated for administration via intrathecal, intravenous orintracranial injection.

One example of a rhabdovirus that was determined to be effective atkilling CNS tumour cell lines while retaining attenuation in normalhuman astrocytes and post-mitotic neurons was Farmington rhabdovirus(FMT). See Example 1 and FIG. 1 for a discussion of the geneticcomponents of FMT. Interestingly, FMT shows little or no sequencehomology to other rhabdoviruses from the six current genera and thus mayconstitute a seventh genus in the Rhabdoviridae. This virus wasdetermined to exhibit reduced neurotoxicity after intracranialadministration (see Example 2 and FIG. 2 ). The FMT virus alsodemonstrated tumour selectivity in vitro (see Example 3 and FIG. 3 ),and safety and efficacy following intracranial or systemicadministration in syngeneic and xenograft mouse models of glioblastoma(see Example 4 and FIG. 4 ).

As discussed above, several viruses, including Maraba (MRB), Farmington(FMT) and Carajas (CRJ), were determined to have potent killing capacityagainst a variety of cancer cell lines from the NC60 cell panel. Theseviruses were also determined to have an ability to eradicate CNS tumourcell lines. These viruses were tested for their safety and efficacy invitro and in vivo.

Previous viruses, such as wild type and attenuated strains of VSV, arealso known to be potent killers of CNS cell lines. However, they arenotably neurotoxic and treatment with such viruses often results inrapid weight loss and paralysis upon intracerebral injection at very lowdoses. Such neurotoxicity prevents application of VSV to treating braincancer.

As illustrated in Example 2 and FIG. 2 , it was determined that, likeVSV, both CRJ and MRB also resulted in neurotoxicity in Balb/C micewithin a period of 2-7 days after administration. However, it wassurprisingly determined that FMT demonstrated no neurotoxicity up to 30days following direct intracranial injection (IC) of 1e6 pfu.

FMT was determined to be able to kill GBM cells at low multiplicities ofinfection, and was determined to possess replication kinetics and largeburst sizes that rivaled those of the highly lytic Maraba virus. FMT wasalso determined to be poorly cytolytic in normal human astrocytes andprimary neurons (see Example 3 and FIG. 3 ). The mechanism of tumourselectivity appears to be independent of interferon signaling, as iscurrently the established mechanism of selectivity governing rhabdovirusbased oncolytic agents. FMT appears to infect normal cells equally totumour cells, but only induces apoptosis in tumour cells. FMT wasdetermined to not trigger caspase 8 in normal cells, even though thereis robust virus protein synthesis. Moreover, FMT's selectivecytotoxicity mechanism rendered the virus non-neurotoxic despite itsstrong ability to block interferon (IFN) production. This indicates thatrhabdovirus infections of the CNS cannot be effectively controlled byinterferon anti-virus defenses when viruses are delivered directly intothe brain. When delivered peripherally, FMT was determined to be asattenuated as previously published engineered VSV deltaM51 or MR MG1strains.

As illustrated in Example 4 and FIG. 4 , the FMT virus may be used totreat human xenograft and immunocompetent syngeneic models by eitherlocal regional or systemic administration. In vivo efficacy in a humanorthotopic U87MG model after a single IC or IV dose of FMT is describedin Example 4. In fact, IV even achieved durable cures. Notably bothmodes of delivery are not only able to treat the primary glioma but areable to effectively and durably treat U87MG spinal metastasis in 100% ofthe animals. Based on these results, it is expected that FMT virus couldbe used to treat other cancers, such as, for example, medulloblastomas.It is expected that FMT virus could be used to treat primary cancers aswell as metastasized cancers, such as to the CNS. Although FMT virusexhibits reduced neurotoxicity and is, for that reason, suitable for usein the treatment of neurological tumors, it should be understood thatthe FMT virus may be used for the treatment of non-neurological cancers.It should also be understood that other viruses according to the presentdisclosure could be used for the treatment of non-neurological cancers.

FMT virus has been demonstrated to induce an anti-tumor immunity. Asillustrated in Example 5 and FIG. 5 , mice previously harboring CT-2Atumors that had been successfully treated with IC FMT virus infusionswere injected for a second time with CT-2A cells directly into thebrain. It was determined that previously cured mice rejected the cells.When, concomitant with FMT virus treatment, cytotoxic T-lymphocytes(CTL) were removed using antibodies directed toward CD8, it wasdetermined that mice stripped of their CD8+ T-cells all eventuallyre-grew the subsequently injected CT-2A cells and failed therapy. Thissuggests that in addition to direct tumour cell lysis and putative othermechanisms of action, the FMT virus induces an anti-tumor immunity whenCTLs are present. Accordingly, it is expected that a virus according tothe present disclosure may be used to induce an immune response in apatient exposed to the virus. The immune response may be, for example,an anti-cancer immune response.

Several groups have also shown impressive efficacy in U87MG model butseldom in a syngeneic immunocompetent GBM model. Moreover, GBM modelsare often treated at predetermined times when animals are still healthyand tumours presumably small. In the examples discussed herein,treatment was commenced 14 days post implantation, which isapproximately 4-7 days before the animals displayed symptoms of theirdisease. In the orthoptopic CT-2A syngeneic GBM model, treatment wascommenced at a stage (19 days post implantation) when animals starteddisplaying overt symptoms of disease. These symptoms include lack ofgrooming, hydrocephaly, and hunched phenotype. This treatment protocolis believed to be particularly relevant to the clinical setting wherepatients are diagnosed and treated after presenting with symptoms. Inthe examples discussed herein, either a single IC dose or 6 IV doses ofFMT was administered. The results demonstrated a similarly significantsurvival profile achieving a significant prolongation in survival andseveral mice in each group (20-30%) were durably cured beyond 100 days.The CT-2A model was chosen because it resulted in an aggressiveinfiltrative tumour and shares proliferative, metabolic, histological,and immunohistochemical profiles observed in human glioblastomamultiforme.

The FMT virus was also demonstrated to not distinguish tumor fromnon-tumor cells via differential infectivity or viral protein production(see Example 6 and FIG. 6 ). The FMT virus is similarly productive innon-cancerous as compared to cancerous cells. Example 6 alsodemonstrated that the genome of wild type FMT virus may be mutated toinclude an additional protein, in the present example the additionalprotein added to the genome of wild type FMT virus was green fluorescentprotein (GFP). The resulting mutated “rec-FMT-GFP virus” wasdemonstrated to block human type I interferon response and productivelyinfect both cancerous and non-cancerous cells.

The FMT virus was also demonstrated to induce cell death in a mannerdependent on the anti-apoptotic threshold of the infected cells, and noton the productivity of the virus infection within the infected cell (seeExample 7 and FIG. 7 ). FMT viral infection of a cell appears toinitiate activation (cleavage) of caspase 8, caspase 9, BH3-interactingdomain and Poly(ADP-ribose) Polymerase in tumor cells.

In summary, FMT is an exemplary oncolytic virus according to the presentdisclosure, and which has been demonstrated to have a high therapeuticindex against human brain cancer cell lines and patient samples invitro, and which has a demonstrated potent efficacy when used to treatpreclinical models of brain cancer. Accordingly, it is expected thatisolated viral particles according to the present disclosure may be usedto treat cancer, such as brain cancer (for example glioblastoma). It isalso expected that isolated viral particles according to the presentdisclosure may be used to induce an immunogenic response, such as ananti-cancer response, in a person administered the virus.

Polynucleotide and Amino Acid Sequences

Polynucleotides comprising nucleic acid sequences (e.g., DNA and RNA)and amino acid (e.g., protein) sequences are provided that may be usedin a variety of methods and techniques known to those skilled in the artof molecular biology. These include isolated, purified, and recombinantforms of the listed sequences and further include complete or partialforms of the listed sequences. Non-limiting uses for amino acidsequences include making antibodies to proteins or peptides comprisingthe disclosed amino acid sequences. Non-limiting uses for thepolynucleotide sequences include making hybridization probes, as primersfor use in the polymerase chain reaction (PCR), for chromosome and genemapping, and the like. Complete or partial amino acid or polynucleotidesequences can be used in such methods and techniques.

The present disclosure features the identification of polynucleotidesequences, including gene sequences and coding nucleic acid sequences,and amino acid sequences. In addition to the sequences expresslyprovided in the accompanying sequence listing, also included arepolynucleotide sequences that are related structurally and/orfunctionally. Also included are polynucleotide sequences that hybridizeunder stringent conditions to any of the polynucleotide sequences in thesequence listing, or a subsequence thereof (e.g., a subsequencecomprising at least 100 contiguous nucleotides). Polynucleotidesequences also include sequences and/or subsequences configured for RNAproduction and/or translation, e.g., mRNA, antisense RNA, sense RNA, RNAsilencing and interference configurations, etc.

Polynucleotide sequences that are substantially identical to thoseprovided in the sequence listing can be used in the compositions andmethods disclosed herein. Substantially identical or substantiallysimilar polynucleotide sequences are defined as polynucleotide sequencesthat are identical, on a nucleotide by nucleotide basis, with at least asubsequence of a reference polynucleotide. Such polynucleotides caninclude, e.g., insertions, deletions, and substitutions relative to anyof those listed in the sequence listing. For example, suchpolynucleotides are typically at least about 70% identical to areference polynucleotide selected from those in the sequence listing, ora subsequence thereof. For example, at least 7 out of 10 nucleotideswithin a window of comparison are identical to the reference sequenceselected. Furthermore, such sequences can be at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or atleast about 99.5%, identical to the reference sequence. Subsequences ofthese polynucleotides can include at least about 5, at least about 10,at least about 15, at least about 20, at least about 25, at least about50, at least about 75, at least about 100, at least about 500, about1000 or more, contiguous nucleotides or complementary subsequences. Suchsubsequences can be, e.g., oligonucleotides, such as syntheticoligonucleotides, isolated oligonucleotides, or full-length genes orcDNAs. Polynucleotide sequences complementary to any of the describedsequences are included.

Amino acid sequences include the amino acid sequences represented in thesequence listing, and subsequences thereof. Also included are amino acidsequences that are highly related structurally and/or functionally. Forexample, in addition to the amino acid sequences in the sequencelisting, amino acid sequences that are substantially identical can beused in the disclosed compositions and methods. Substantially identicalor substantially similar amino acid sequences are defined as amino acidsequences that are identical, on an amino acid by amino acid basis, withat least a subsequence of a reference amino acid sequence. Such aminoacid sequences can include, e.g., insertions, deletions, andsubstitutions relative to any of the amino acid sequences in thesequence listing. For example, such amino acids are typically at leastabout 70% identical to a reference amino acid sequence, or a subsequencethereof. For example, at least 7 out of 10 amino acids within a windowof comparison are identical to the reference amino acid sequenceselected. Frequently, such amino acid sequences are at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or at least about 99.5%, identical to the reference sequence.Subsequences of the amino acid sequences can include at least about 5,at least about 10, at least about 15, at least about 20, at least about25, at least about 50, at least about 75, at least about 100, at leastabout 500, about 1000 or more, contiguous amino acids. Conservativevariants of amino acid sequences or subsequences are also possible.Amino acid sequences can be immunogenic, enzymatically active,enzymatically inactive, and the like.

Where the polynucleotide sequences are translated to form a polypeptideor subsequence of a polypeptide, nucleotide changes can result in eitherconservative or non-conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving functionally similar side chains. Conservative substitutiontables providing functionally similar amino acids are well known in theart. Table 1 sets forth examples of six groups containing amino acidsthat are “conservative substitutions” for one another. Otherconservative substitution charts are available in the art, and can beused in a similar manner.

TABLE 1 Conservative Substitution Group 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid(E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

One of skill in the art will appreciate that many conservativesubstitutions yield functionally identical constructs. For example, asdiscussed above, owing to the degeneracy of the genetic code, “silentsubstitutions” (i.e., substitutions in a polynucleotide sequence whichdo not result in an alteration in an encoded polypeptide) are an impliedfeature of every polynucleotide sequence which encodes an amino acid.Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence (e.g., about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10% or more) are substituted with different amino acidswith highly similar properties, are also readily identified as beinghighly similar to a disclosed construct. Such conservative variations ofeach disclosed sequence are also contemplated.

Methods for obtaining conservative variants, as well as more divergentversions of the polynucleotide and amino acid sequences, are widelyknown in the art. In addition to naturally occurring homologues whichcan be obtained, e.g., by screening genomic or expression librariesaccording to any of a variety of well-established protocols, see, e.g.,Ausubel et al. Current Protocols in Molecular Biology (supplementedthrough 2004) John Wiley & Sons, New York (“Ausubel”); Sambrook et al.Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”), andBerger and Kimmel Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif.(“Berger”), additional variants can be produced by any of a variety ofmutagenesis procedures. Many such procedures are known in the art,including site directed mutagenesis, oligonucleotide-directedmutagenesis, and many others. For example, site directed mutagenesis isdescribed, e.g., in Smith (1985) “In vitro mutagenesis” Ann. Rev. Genet.19:423-462, and references therein, Botstein & Shortle (1985)“Strategies and applications of in vitro mutagenesis” Science229:1193-1201; and Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7. Oligonucleotide-directed mutagenesis is described, e.g., inZoller & Smith (1982) “Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment” Nucleic Acids Res.10:6487-6500). Mutagenesis using modified bases is described e.g., inKunkel (1985) “Rapid and efficient site-specific mutagenesis withoutphenotypic selection” Proc. Natl. Acad. Sci. USA 82:488-492, and Tayloret al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787. Mutagenesis using gapped duplex DNA isdescribed, e.g., in Kramer et al. (1984) “The gapped duplex DNA approachto oligonucleotide-directed mutation construction” Nucl. Acids Res. 12:9441-9460). Point mismatch mutagenesis is described, e.g., by Kramer etal. (1984) “Point Mismatch Repair” Cell 38:879-887). Double-strand breakmutagenesis is described, e.g., in Mandecki (1986)“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177-7181, and in Arnold (1993) “Protein engineeringfor unusual environments” Current Opinion in Biotechnology 4:450-455).Mutagenesis using repair-deficient host strains is described, e.g., inCarter et al. (1985) “Improved oligonucleotide site-directed mutagenesisusing M13 vectors” Nucl. Acids Res. 13: 4431-4443. Mutagenesis by totalgene synthesis is described e.g., by Nambiar et al. (1984) “Totalsynthesis and cloning of a gene coding for the ribonuclease S protein”Science 223: 1299-1301. DNA shuffling is described, e.g., by Stemmer(1994) “Rapid evolution of a protein in vitro by DNA shuffling” Nature370:389-391, and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution,” Proc.Natl. Acad. Sci. USA 91:10747-10751.

Many of the above methods are further described in Methods in EnzymologyVolume 154, which also describes useful controls for trouble-shootingproblems with various mutagenesis methods. Kits for mutagenesis, libraryconstruction and other diversity generation methods are alsocommercially available. For example, kits are available from, e.g.,Amersham International plc (Piscataway, N.J.) (e.g., using the Ecksteinmethod above), Bio/Can Scientific (Mississauga, Ontario, CANADA),Bio-Rad (Hercules, Calif.) (e.g., using the Kunkel method describedabove), Boehringer Mannheim Corp. (Ridgefield, Conn.), ClonetechLaboratories of BD Biosciences (Palo Alto, Calif.), DNA Technologies(Gaithersburg, Md.), Epicentre Technologies (Madison, Wis.) (e.g., the 5prime 3 prime kit); Genpak Inc. (Stony Brook, N.Y.), Lemargo Inc(Toronto, CANADA), Invitrogen Life Technologies (Carlsbad, Calif.), NewEngland Biolabs (Beverly, Mass.), Pharmacia Biotech (Peapack, N.J.),Promega Corp. (Madison, Wis.), QBiogene (Carlsbad, Calif.), andStratagene (La Jolla, Calif.) (e.g., QuickChange™ site-directedmutagenesis kit and Chameleon™ double-stranded, site-directedmutagenesis kit).

Determining Sequence Relationships

Similar sequences can be objectively determined by any number ofmethods, e.g., percent identity, hybridization, immunologically, and thelike. A variety of methods for determining relationships between two ormore sequences (e.g., identity, similarity and/or homology) areavailable and well known in the art. Methods include manual alignment,computer assisted sequence alignment, and combinations thereof, forexample. A number of algorithms (which are generally computerimplemented) for performing sequence alignment are widely available orcan be produced by one of skill. These methods include, e.g., the localhomology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482;the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443; the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. (USA) 85:2444; and/or by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Dr., Madison, Wis.).

For example, software for performing sequence identity (and sequencesimilarity) analysis using the BLAST algorithm is described in Altschulet al. (1990) J. Mol. Biol. 215:403-410. This software is publiclyavailable, e.g., through the National Center for BiotechnologyInformation on the internet at ncbi.nlm.nih.gov. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (N) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP (BLASTProtein) program uses as defaults a wordlength (N) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff(1989) Proc. Natl. Acad. Sci. USA 89:10915).

Additionally, the BLAST algorithm performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul (1993)Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (p(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence(and, therefore, in this context, homologous) if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, or less than about 0.01, and oreven less than about 0.001.

Another example of a sequence alignment algorithm is PILEUP, whichcreates a multiple sequence alignment from a group of related sequencesusing progressive, pairwise alignments. It can also plot a tree showingthe clustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle(1987) J. Mol. Evol. 35:351-360. The method used is similar to themethod described by Higgins & Sharp (1989) CABIOS5:151-153. The programcan align, e.g., up to 300 sequences of a maximum length of 5,000letters. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster can then be aligned to the next mostrelated sequence or cluster of aligned sequences. Two clusters ofsequences can be aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program can also be used toplot a dendogram or tree representation of clustering relationships. Theprogram is run by designating specific sequences and their amino acid ornucleotide coordinates for regions of sequence comparison.

An additional example of an algorithm that is suitable for multiple DNA,or amino acid, sequence alignments is the CLUSTALW program (Thompson, J.D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performsmultiple pairwise comparisons between groups of sequences and assemblesthem into a multiple alignment based on homology. Gap open and Gapextension penalties can be, e.g., 10 and 0.05 respectively. For aminoacid alignments, the BLOSUM algorithm can be used as a protein weightmatrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.USA 89: 10915-10919.

Polynucleotide hybridization similarity can also be evaluated byhybridization between single stranded (or single stranded regions of)nucleic acids with complementary or partially complementarypolynucleotide sequences. Hybridization is a measure of the physicalassociation between nucleic acids, typically, in solution, or with oneof the nucleic acid strands immobilized on a solid support, e.g., amembrane, a bead, a chip, a filter, etc. Nucleic acid hybridizationoccurs based on a variety of well characterized physico-chemical forces,such as hydrogen bonding, solvent exclusion, base stacking, and thelike. Numerous protocols for nucleic acid hybridization are well knownin the art. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, part I,chapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays,” (Elsevier, N.Y.), as well as in Ausubel etal. Current Protocols in Molecular Biology (supplemented through 2004)John Wiley & Sons, New York (“Ausubel”); Sambrook et al. MolecularCloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”), and Berger andKimmel Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (“Berger”). Hames andHiggins (1995) Gene Probes 1, IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 1) and Hames and Higgins (1995) GeneProbes 2, IRL Press at Oxford University Press, Oxford, England (Hamesand Higgins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

Conditions suitable for obtaining hybridization, including differentialhybridization, are selected according to the theoretical meltingtemperature (Tm) between complementary and partially complementarynucleic acids. Under a given set of conditions, e.g., solventcomposition, ionic strength, etc., the. Tm is the temperature at whichthe duplex between the hybridizing nucleic acid strands is 50%denatured. That is, the Tm corresponds to the temperature correspondingto the midpoint in transition from helix to random coil; it depends onthe length of the polynucleotides, nucleotide composition, and ionicstrength, for long stretches of nucleotides.

After hybridization, unhybridized nucleic acids can be removed by aseries of washes, the stringency of which can be adjusted depending uponthe desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the T.sub.m) lower the background signal, typicallywith primarily the specific signal remaining, See, also, Rapley, R. andWalker, J. M. eds., Molecular Biomethods Handbook (Humana Press, Inc.1998).

“Stringent hybridization wash conditions” or “stringent conditions” inthe context of nucleic acid hybridization experiments, such as Southernand northern hybridizations, are sequence dependent, and are differentunder different environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993), supra, and inHames and Higgins 1 and Hames and Higgins 2, supra.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 2×SSC, 50%formamide at 42° C., with the hybridization being carried out overnight(e.g., for approximately 20 hours). An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook,supra for a description of SSC buffer). Often, the wash determining thestringency is preceded by a low stringency wash to remove signal due toresidual unhybridized probe. An example low stringency wash is 2×SSC atroom temperature (e.g., 20° C. for 15 minutes).

In general, a signal to noise ratio of at least 2.5×-5× (and typicallyhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Detection of at least stringent hybridization between two sequencesindicates relatively strong structural similarity to those provided inthe sequence listings herein.

Generally, “highly stringent” hybridization and wash conditions areselected to be about 5° C. or less lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength and pH (asnoted below, highly stringent conditions can also be referred to incomparative terms). Target sequences that are closely related oridentical to the nucleotide sequence of interest (e.g., “probe”) can beidentified under stringent or highly stringent conditions. Lowerstringency conditions are appropriate for sequences that are lesscomplementary.

For example, in determining stringent or highly stringent hybridization(or even more stringent hybridization) and wash conditions, thestringency of the hybridization and wash conditions is graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration, and/or increasing theconcentration of organic solvents, such as formamide, in thehybridization or wash), until a selected set of criteria are met. Forexample, the stringency of the hybridization and wash conditions isgradually increased until a probe comprising one or more of the presentpolynucleotide sequences, or a subsequence thereof, and/or complementarypolynucleotide sequences thereof, binds to a perfectly matchedcomplementary target, with a signal to noise ratio that is at least2.5×, and optionally 5×, or 10×, or 100× or more, as high as thatobserved for hybridization of the probe to an unmatched target, asdesired.

Using subsequences derived from the nucleic acids listed in the sequencelisting, novel target nucleic acids can be obtained; such target nucleicacids are also a feature of the invention. For example, such targetnucleic acids include sequences that hybridize under stringentconditions to an oligonucleotide probe that corresponds to a uniquesubsequence of any of the polynucleotides in the sequence listing, or acomplementary sequence thereof; the probe optionally encodes a uniquesubsequence in any of the amino acid sequences of the sequence listing.

For example, hybridization conditions are chosen under which a targetoligonucleotide that is perfectly complementary to the oligonucleotideprobe hybridizes to the probe with at least about a 5-10× higher signalto noise ratio than for hybridization of the target oligonucleotide to anegative control non-complimentary nucleic acid. Higher ratios of signalto noise can be achieved by increasing the stringency of thehybridization conditions such that ratios of about 15×, 20×, 30×, 50× ormore are obtained. The particular signal will depend on the label usedin the relevant assay, e.g., a fluorescent label, a calorimetric label,a radioactive label, or the like.

Vectors, Promoters and Expression Systems

Polynucleotide sequences of the present disclosure can be in any of avariety of forms, e.g., expression cassettes, vectors, plasmids, viralparticles, or linear nucleic acid sequences. For example, vectors,plasmids, cosmids, bacterial artificial chromosomes (BACs), YACs (yeastartificial chromosomes), phage, viruses and nucleic acid segments cancomprise the present nucleic acid sequences or subsequences thereof.These nucleic acid constructs can further include promoters, enhancers,polylinkers, regulatory genes, etc. Thus, the present disclosure alsorelates, e.g., to vectors comprising the polynucleotides disclosedherein, host cells that incorporate these vectors, and the production ofthe various disclosed polypeptides (including those in the sequencelisting) by recombinant techniques.

In accordance with these aspects, the vector may be, for example, aplasmid vector, a single or double-stranded phage vector, or a single ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells as polynucleotides, preferably DNA, by well known techniquesfor introducing DNA and RNA into cells. The vectors, in the case ofphage and viral vectors, also may be and preferably are introduced intocells as packaged or encapsidated virus by well known techniques forinfection and transduction. Viral vectors may be replication competentor replication defective. In the latter case, viral propagationgenerally will occur only in complementing host cells.

In some examples, vectors include those useful for expression ofpolynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host, operably linked to the polynucleotide to beexpressed. Appropriate trans-acting factors are supplied by the host,supplied by a complementing vector or supplied by the vector itself uponintroduction into the host.

In certain examples in this regard, the vectors provide for proteinexpression. Such preferred expression may be inducible expression,temporally limited expression, or expression restricted to predominantlycertain types of cells, or any combination of the above. Someembodiments of inducible vectors can be induced for expression byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives. A variety of vectors suitable to this aspect,including constitutive and inducible expression vectors for use inprokaryotic and eukaryotic hosts, are well known and employed routinelyby those of skill in the art. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as rhabdoviruses, baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids andbinaries used for Agrobacterium-mediated transformations.

Vectors can include a selectable marker and a reporter gene. For ease ofobtaining sufficient quantities of vector, a bacterial origin thatallows replication in E. coli can be used. The following vectors, whichare commercially available, are provided by way of example. Amongvectors preferred for use in bacteria are pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Useful plant binary vectors include BIN19 and itsderivatives available from Clontech. These vectors are listed solely byway of illustration of the many commercially available and well-knownvectors that are available to those of skill in the art. It will beappreciated that any other plasmid or vector suitable for, for example,introduction, maintenance, propagation or expression of one or morepolynucleotides and/or polypeptides as provided in the present sequencelisting, including variants thereof as described, in a host may be used.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, aribosome-binding site for translation when the construct encodes apolypeptide. The coding portion of the mature transcripts expressed bythe constructs will include a translation-initiating AUG at thebeginning and a termination codon appropriately positioned at the end ofthe polypeptide to be translated. In addition, the constructs maycontain control regions that regulate as well as engender expression.Generally, in accordance with many commonly practiced procedures, suchregions will operate by controlling transcription, such as transcriptionfactors, repressor binding sites and termination signals, among others.For secretion of a translated protein into the lumen of the endoplasmicreticulum, into the periplasmic space or into the extracellularenvironment, appropriate secretion signals may be incorporated into theexpressed polypeptide. These signals may be endogenous to thepolypeptide or they may be heterologous signals.

Transcription of the DNA (e.g., encoding the polypeptides) of thepresent invention by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at bp 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.Additional enhancers useful in the invention to increase transcriptionof the introduced DNA segment, include, inter alia, viral enhancers likethose within the 35S promoter, as shown by Odell et al., Plant Mol.Biol. 10:263-72 (1988), and an enhancer from an opine gene as describedby Fromm et al., Plant Cell 1:977 (1989). The enhancer may affect thetissue-specificity and/or temporal specificity of expression ofsequences included in the vector.

Termination regions also facilitate effective expression by endingtranscription at appropriate points. Useful terminators include, but arenot limited to, pinII (see An et al., Plant Cell 1(1):115-122 (1989)),glb1 (see Genbank Accession #L22345), gz (see gzw64a terminator, GenbankAccession #S78780), and the nos terminator from Agrobacterium. Thetermination region can be native with the promoter nucleotide sequence,can be native with the DNA sequence of interest, or can be derived fromanother source. For example, other convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also: Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

Among known eukaryotic promoters suitable for generalized expression arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), metallothionein promoters, suchas the mouse metallothionein-I promoter and various plant promoters,such as globulin-1. The native promoters of the polynucleotide sequenceslisting in the sequence listing may also be used. Representatives ofprokaryotic promoters include the phage lambda PL promoter, the E. colilac, trp and tac promoters to name just a few of the well-knownpromoters.

Isolated or recombinant viruses, virus infected cells, or cellsincluding one or more portions of the present polynucleotide sequencesand/or expressing one or more portions of the present amino acidsequences are also contemplated.

A polynucleotide, optionally encoding the heterologous structuralsequence of an amino acid sequence as disclosed, generally will beinserted into a vector using standard techniques so that it is operablylinked to a promoter for expression. Operably linked, as used herein,includes reference to a functional linkage between a promoter and asecond sequence, wherein the promoter sequence initiates and mediatestranscription of the DNA corresponding to the second sequence.Generally, operably linked means that the polynucleotide sequence beinglinked is contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. When thepolynucleotide is intended for expression of a polypeptide, thepolynucleotide will be positioned so that the transcription start siteis located appropriately 5′ to a ribosome binding site. Theribosome-binding site will be 5′ to the AUG that initiates translationof the polypeptide to be expressed. Generally, there will be no otheropen reading frames that begin with an initiation codon, usually AUG,and lie between the ribosome binding site and the initiation codon.Also, generally, there will be a translation stop codon at the end ofthe polypeptide and there will be a polyadenylation signal in constructsfor use in eukaryotic hosts. Transcription termination signalsappropriately disposed at the 3′ end of the transcribed region may alsobe included in the polynucleotide construct.

For nucleic acid constructs designed to express a polypeptide, theexpression cassettes can additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example: EMCVleader (Encephalomyocarditis 5′ noncoding region), Elroy-Stein et al.(1989) Proc. Nat. Acad. Sci. USA 86:6126-6130; potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMVleader (Maize Dwarf Mosaic Virus), Virology 154:9-20; humanimmunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991)Nature 353:90-94; untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV), Gallie et al. (1989)Molecular Biology of RNA, pages 237-256; and maize chlorotic mottlevirus leader (MCMV) Lommel et al. (1991) Virology 81:382-385. See alsoDella-Cioppa et al. (1987) Plant Physiology 84:965-968. The cassette canalso contain sequences that enhance translation and/or mRNA stabilitysuch as introns. The expression cassette can also include, at the 3′terminus of the isolated nucleotide sequence of interest, atranslational termination region.

In those instances where it is desirable to have the expressed productof the polynucleotide sequence directed to a particular organelle orsecreted at the cell's surface the expression cassette can furthercomprise a coding sequence for a transit peptide. Such transit peptidesare well known in the art and include, but are not limited to: thetransit peptide for the acyl carrier protein, the small subunit ofRUBISCO, plant EPSP synthase, and the like.

In making an expression cassette, the various DNA fragments can bemanipulated so as to provide for the polynucleotide sequences in theproper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers can be employed to join DNAfragments or other manipulations can be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction digests, annealing, and resubstitutions suchas transitions and transversions, can be employed.

Introduction of a construct into a host cell can be effected by calciumphosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., Basic Methods in Molecular Biology, (1986) andSambrook et al., Molecular Cloning—A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS and Bowes melanoma cells; and plantcells.

The host cells can be cultured in conventional nutrient media, which maybe modified as appropriate for, inter alia, activating promoters,selecting transformants or amplifying genes. Culture conditions, such astemperature, pH and the like, previously used with the host cellselected for expression generally will be suitable for expression ofnucleic acids and/or polypeptides, as will be apparent to those of skillin the art. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the polynucleotides disclosed herein.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period. Cells typically then are harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. Microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents; such methods are well known to those skilled inthe art.

Compositions and methods of the present disclosure can includeadministering the polynucleotides and/or amino acids as provided herein.For example, treatments for glioblastoma can include administering oneor more of the polynucleotides and/or amino acids. The one or morepolynucleotides and/or amino acids may be in an isolated form or may bepart of a composition, including a viral particle. In variousembodiments, the administering can take the following forms:intradermal, transdermal, parenteral, intravascular, intravenous,intramuscular, intranasal, subcutaneous, regional, percutaneous,intratracheal, intraperitoneal, intraarterial, intravesical,intratumoral, inhalation, perfusion, lavage, direct injection,alimentary, oral, or intracranial administration.

Materials and Methods

Cell lines: U87MG, U343, U373, SF268, SF265, SF539, SNB19, SNB75, Vero,U118, normal human astrocytes (NHA), CT-2A, DBT, GL261 and human GM38primary fibroblasts (National Institute of General Medical SciencesMutant Cell Repository, Camden, NJ) were propagated in Dulbecco'smodified Eagle's medium (Hyclone, Logan, UT) supplemented with 10% fetalcalf serum (Cansera, Etobicoke, Ontario, Canada).

Viability Assays: The indicated cell lines were plated at a density of10 000 cells/well into 96 well plates. The next day cells were infectedwith the rMarabaWT, or FMT at various multiplicity of infections(0.0001-10 pfu/cell). Following a 48 hour incubation Alamar Blue(Resazurin sodium salt (Sigma-Aldrich)) was added to a finalconcentration of 20 μg/ml. After a 6 hour incubation the absorbance wasread at a wavelength of 573 nm.

Plaque assays: Vero cells were plated at a density of 5e5 cells per/wellof a 6 well dish. The next day 100 l of serial viral dilutions wereprepared and added for 1 hour to Vero cells. After viral adsorption 2 mlof agarose overlay was added (1:1 1% agarose: 2×DMEM and 20% FCS).Plaques were counted the following day.

Interferon bioassay: PC-3 cells were infected with rMarabaWT, ΔM51 orFMT, at a multiplicity of infection of 3 pfu/cell for 24 hours. Thefollowing day supernatant was acid neutralized with 0.25N HCl overnightat 4° C. followed by the addition of 0.25 NaOH to adjust the pH to 7.Vero cells were incubated with the neutralized supernatant for 24 hoursand subsequently infected rMaraba WT with a multiplicity of infectionranging from 0.0001 to 100 pfu/cell. Any interferon secreted by the PC-3cells in response to Maraba or the attenuated mutants would subsequentlyprotect the Vero cells from infection with Maraba. After 24 hours,survival was quantitated using a crystal violet assay. Briefly cellswere incubated with 1% crystal violet solution, washed, dried,resuspended in 1% SDS and read at a wavelength of 595 nm.

Determination of in vivo toxicity: For the intracranial (IC) route ofadministration (roa), groups of 6-8 week old female BALB/c mice(n=5/group) were given a single IC infusion of the indicated viruses inlog increments per group ranging from 10²-10⁷ pfu. For the intravenous(IV) roa, groups of five 6-8 week old female BALB/c mice were given asingle IV injection of the indicated viruses into the tail vein, in halflog increments per group ranging from 3×10⁶-3×10⁹ pfu, diluted into 100μL per injection. Following IC or IV injections, mice were monitoreddaily for signs of distress including weight loss, piloerection,hind-limb paralysis and respiratory distress. The median lethal dose(LD50) was calculated using the Spearman Karber method, while themaximal tolerable dose (MTD) was denoted as the highest dose notresulting in a single animal death.

Imaging glioblastoma in an animal model: U87MG and CT2A cells wereadapted for bioluminescent imaging by transducing with lentiviruscontaining firefly luciferase (FLUC) and transfecting FLUC plasmidrespectively. U87MG FLUC and CT2A FLUC cells were injected IC into CD1nude and C57BL/6 respectively. Animals with FLUC expressing tumours weremonitored for tumour progression using the live imaging IVIS Xenogen 200system after an IP injection of luciferin (Gold Biotechnology Inc). Theanimals were monitored for signs of distress including survival, weightloss, morbidity, piloerection, hind-limb paralysis and respiratorydistress.

Mouse syngeneic glioblastoma tumour models: Brain tumours wereestablished by a single stereotactic injection with CT-2A mouse gliomacells into 6-8 week old C57BL/6 animals. Five days post injection. OnDay 19 C57BL/6 mice bearing CT-2A tumours were IV treated with 6 dosesof FMT (5×10⁸ pfu/dose thrice weekly) or injected stereotactically withFMT (2×10⁷ in a volume of 50 μl) using an infusion pump (rate=3 μl/min).Some 057Bl/6 animals were sacrificed at day 19 and images were capturedon a Nikon dissecting microscope. The remaining animals were monitoredfor survival.

Human glioblastoma xenograft model: Human ovarian U87MG cells wereadapted for bioluminescent imaging at which time 1e6 U87MG cells wereinjected IC into 6-8 week old athymic CD-1 nude mice. Untreated CD-1animals develop tumours at about day 15-21. Mice were either treatedwith a single intravenous (tail vein) injection performed on day 14 withFMT (5×10⁸), or treated IC with the same virus at a dose of 2×10⁷ pfu.Animals were monitored for survival and for signs of distress includingweight loss, morbidity, piloerection, hind-limb paralysis andrespiratory distress. Tumour imaging was captured with a Xenogen 200IVIS system (Caliper LS, USA).

Rotorod: Balb/C mice were tested for motor function/performance on arotating rod apparatus prior to IC viral administration. Mice wereplaced on a rotorod for 3 trials per day for 4 consecutive days. Afterallowing the animals 0.5 min to adjust to the apparatus, the rod wasaccelerated in a linear fashion 0.1 rpm/s. Latency to fall was measuredin minutes. The animals were divided into groups of 3. Motor functionone week post surgery in uninjected (Naïve), PBS and FMT IC treatedanimals. Standard error of the mean was calculated.

Nucleic Acid Sequencing: FMT sequencing was performed at the OntarioInstitute for Cancer research (Toronto, Canada) on FMT cDNA which wasgenerated using a shotgun approach with random hexamers on trizolextracted and RNeasy purified FMT RNA.

Protein sequencing: FMT virus was amplified in Vero cells to high titer(˜10¹¹ pfu/mL), purified, and lysed with 5× Laemmli sample buffer (60 mMTris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01%bromophenol blue) and separated on 12% SDS-PAGE gels. Replicate gelswere stained with either coomasie blue or silver, and nine bands wereextracted for peptide sequencing.

Manufacturing and rescuing recombinant FMT virus: Recombinant FMT wasproduced as described recently for Maraba virus25. Briefly, FMT viruscomplementary DNA (cDNA) was amplified in three separate RT-PCRreactions yielding overlapping fragments that were stitched togetherusing internal restriction sites. The full length ˜11 Kb cDNA was thencloned into a modified LC-KAN vector (Lucigen, Middleton, WI) carrying aT7 promoter upstream of the 5′-antigenomic leader sequence andimmediately downstream of the 3′-terminator a modified hepatitis deltavirus ribozyme and T7 polymerase termination signal sequence. A549 lungcarcinoma cells seeded at 3.0×10⁵ cells/well in 6-well plates wereinfected 24 hr later with vaccinia virus (moi=10) expressing the T7 RNApolymerase37 in OptiMeM medium for 1.5 hours. Following removal of thevaccinia virus, each well was transfected with LC-KAN FMT (2 μg)together with pCl-Neo constructs encoding for FMT “N” (1 μg), “P” (1.25μg), and L (0.25 μg) with lipofectamine 2000 (5 μl per well) accordingto the manufacturer's instructions. The transfection reagent was removed5 hr later and replaced with DMEM containing 10% HI-FBS. At 48 hoursfollowing the transfection, medium was collected (pooled from twoplates), filtered (0.2 μm) to remove contaminating vaccinia virus, and 1ml was used to infect SNB19 glioblastoma cells in each well of a 6-wellplate. Cytopathic effects and GFP expression visible 24-48 hours laterwere indicative of a successful rescue. Recombinant FMT underwent threerounds of plaque purification (on SNB19 cells), before scale up,purification on sucrose cushion, and resuspension in PBS containing 15%glucose.

Phylogenetic Analysis: Phylogenetic relationships between rhabdovirusesbased on a Muscle alignment of L protein amino acid sequences, and usingthe paramyxovirus Measles Edmonston strain as the outgroup. The tree wasgenerated by the neighbor-joining method and bootstrap values (indicatedfor each branch node) were estimated using 1000 tree replicas. Branchlengths are proportional to genetic distances. The scale bar correspondsto substitutions per amino acid site.

Immunoblotting: Cells were lysed (50 mM Tris-HCl; 150 mM NaCl; 1% TritonX-100; 1% SDS) and protein quantified using the Lowry assay (Bio-Rad).Total cell lysates were prepared in SDS sample buffer, and 5-50 μg oftotal protein was separated by SDS-PAGE on Bis-Tris gels (ranging from8-15%) and transferred to nitrocellulose or PVDF membranes. Membraneswere probed with primary antibodies diluted in 5% skim milk powder (SMP)or 5% Bovine Serum Albumen (BSA) overnight at 4 deg C., followed byhorse radish peroxidase-conjugated secondary antibodies diluted in 5%SMP for 1 hr at room temperature. The following primary antibodies wereused: rabbit anti-PARP (Cell Signaling, 9542); mouse anti-caspase 3(Cell Signaling, 9668); mouse anti-caspase 8 (Enzo Life Sciences, 12F5);rabbit anti-caspase 9 (Cell Signaling, 9502); rabbit anti-BID (CellSignaling, 2002); mouse anti-GAPDH (R&D Systems). A polylonal anti-FMTantibody was generated in rabbits (Capralogics Inc.) using purified,UV-inactivated FMT virus. Protein bands were visualized usingSuperSignal West Pico Chemiluminescent Substrate System (PierceBiotechnology).

CD8+ T cell depletion: The brains of 7 week old C57BL/6 mice werestereotactically implanted with 2e5 CT2A cells that express fireflyluciferase (CT2Afluc). Mice were treated with 2e7 PFU of Farmingtonvirus at the site of tumour implantation 7 days later. For CD8+ T celldepletion studies, 200 μg of anti-mouse CD8 antibody (clone 2.43) wasinjected intraperitoneally (IP) one day prior to virus treatment and 100μg on the day of treatment. A maintenance dose of 100 μg every 3 dayswas given for the following 2 weeks.

Statistics: For Kaplan Meier plots, survival plots were compared usingMantel-Cox Log rank analysis (Graphpad Prism).

EXAMPLES Example 1: Farmington Virus is not a Vesiculovirus

The full-length genomic sequence for FMT was determined. The sequence ofthe complementary DNA (cDNA) polynucleotide produced by FMT is shown inSEQ ID NO: 1. The RNA polynucleotide of FMT is shown in SEQ ID NO: 2.Five putative open reading frames were identified in the genomicsequence. Additional ORFs may be present in the virus that have not yetbeen identified. The sequences of the corresponding proteins are shownin SEQ ID NOs: 3, 4, 5, 6 and 7, and the encoding DNA sequences areshown in SEQ ID NOs: 8, 9, 10, 11 and 12, respectively. Phylogeneticanalysis of the full-length genomic sequence was performed by aligningthe amino acid sequence of the putative FMT L protein to the L proteinsequences of representative members from the 6 genera of Rhabdoviridae(FIG. 1 ). The alignment demonstrated that FMT did not appear to belongto the current 6 genera schema of the Rhabdoviridae family. FMT virusappears to be more divergent from the currently known rhabdoviruses.While we did detect some sequence homology (˜50% identity) between ashort segment of the L protein of FMT and lettuce necrosis yellow virus,we were unable to detect any homology of the 4 remaining FMT putativeopen reading frames (ORFs) (N, P, G, M) to any sequences in the NCBIdatabase. This suggests that FMT, which was originally classifiedincorrectly as a vesiculovirus (Tesh et al. Emerging Infect. Dis. 2002),may in fact constitute the type member of a new genus within theRhabdoviridae family (FIG. 1 ; and see Table 2 and accompanying SequenceListing file).

TABLE 2 Description of Sequences. SEQ ID Farmington rhabdovirus - CDNAproduced by the FMT NO. 1 DNA rhabdovirus SEQ ID Farmingtonrhabdovirus - NO. 2 RNA SEQ ID Farmington rhabdovirus The promoter is atposition NO. 3 ORF 1 134 to 149 and the encoding sequence is atpositions 206 to 1444 of SEQ ID NO: 1 SEQ ID Farmington rhabdovirus Thepromoter is at position NO. 4 ORF 2 1562 to 1578 and the encodingsequence is at positions 1640 to 2590 of SEQ ID NO: 1 SEQ ID Farmingtonrhabdovirus The promoter is at positions NO. 5 ORF 3 2799 to 2813 andthe encoding sequence is at positions 2894 to 3340 of SEQ ID NO: 1 SEQID Farmington rhabdovirus The promoter is at positions NO. 6 ORF 4 3457to 3469 and the encoding sequence is at positions 3603 to 5717 of SEQ IDNO: 1 SEQ ID Farmington rhabdovirus The promoter is at positions NO. 7ORF 5 5766 to 5780 and the encoding sequence is at positions 5832 to12221 of SEQ ID NO: 1

SEQ ID NO: 3 is encoded by SEQ ID NO: 8 (i.e. the encoding sequence ofpositions 206 to 1444 of SEQ ID NO: 1). SEQ ID NO: 4 is encoded by SEQID NO: 9 (i.e. the encoding sequence of positions 1640 to 2590 of SEQ IDNO: 1). SEQ ID NO: 5 is encoded by SEQ ID NO: 10 (i.e. the encodingsequence of positions 2894 to 3340 of SEQ ID NO: 1). SEQ ID NO: 6 isencoded by SEQ ID NO: 11 (i.e. the encoding sequence of positions 3603to 5717 of SEQ ID NO: 1). SEQ ID NO: 7 is encoded by SEQ ID NO: 12 (i.e.the encoding sequence of positions 5832 to 12221 of SEQ ID NO: 1).

FIG. 1 shows A) Schematic of where FMT was first isolated in 1969. B)Amino acid Blast alignment of 5 FMT ORFs reveals no sequence homology toany other sequences in the database except for the L protein which is˜45% similar to plant rhabdovirus: Lettuce Necrotic yellow virus (LNYV).C) Commassie stained SDS PAGE gel of FMT virus showing 4 of the 5predicted FMT ORF proteins. These bands were excised from the gel andtheir identity confirmed through protein sequencing by tandem massspectrometry. D) Phylogenic tree derived from the amino acid sequencesof the polymerase genes of various rhabdoviruses. The Measlesparamyxovirus was included as a non-family control. All previousoncolytic rhabdoviruses have been identified from the vesiculovirusgenus (VSV, Maraba). Unexpectedly, Farmington virus appears to clusterwith the plant infecting cytorhabdoviruses. E) We have built arecombinant system for the FMT platform that allows us complete controlof the genetic make-up of the virus. We show here the generation of afully replicative GFP expressing FMT strain as an example. F) Electronmicrogram of FMT bullet shaped virion measuring 55 nm×150 nm. (adaptedfrom Tesh et al. Emerging Infect. Dis. 2002).

Example 2: Farmington Virus does not Demonstrate Neurotoxicity

FMT demonstrated profound attenuation in non-transformed cells in vitro.To ascertain whether the observed attenuation in vitro translates tosafety in vivo, we injected stereotaxically into the right striatum ofthe brain increasing doses of rhabdoviruses and monitored for signs ofneurotoxicity (including weight loss, piloerection, hind leg paralysis,morbidity and mortality). FMT had an LD₅₀ of approximately 1×10⁹ incontrast the other rhabdoviruses displayed LD₅₀ of approximately 10².(FIG. 2A). The Maximum Tolerated Dose (MTD) was determined to be thehighest dose not resulting in durable morbidity. (FIG. 2A). For FMT theMTD was approximately 1×10^(7.5) pfu while for the other rhabdovirusesthe LD₅₀ occurred at such a low dose of virus the MTD was impossible todetermine. These animals displayed clinical signs of a CNS infectionwith rapid and progressive weight loss, hind leg paralysis and hadsignificant titres of virus in their brain just prior to death (data notshown).

Although we found no acute neurotoxicity from IC treatment with FMT, wewished to assess the cognitive and motor function of the mice severaldays after virus infection. Therefore, we assessed the motor functionbefore and after treatment with these wild-type FMT virus using arotorod apparatus (FIG. 2B). Specifically, we measured the latency ofthese animals to fall from a slowly accelerating rod. We show that thereis no significant difference in the latency to fall between themock-infected animals or virus infected animals, 1 week prior and 1 weekpost injection (FIG. 2B).

In addition to intracranial toxicity, we evaluated the toxicity of FMTwhen administered intravenously (IV) in immunocompetent mice withescalating doses of virus (FIG. 2C). FMT is well tolerated IV and neverreaches an LD₅₀ even at our highest dose 3×10⁹ pfu which is comparableto an attenuated version of Maraba we described in Brun, J. et al.Identification of Genetically Modified Maraba Virus as an OncolyticRhabdovirus. Mol Ther 18, 1440 (2010). FMT animals IV dosed at greaterthan 3×10⁸ pfu displayed transient weight loss and moderatepiloerection, which resolved 5-7 days post treatment (data not shown).No detectable titres were observed from brain homogenates taken from FMTtreated animals 3 months post IV treatment (FIG. 2D). In addition,following administration of high doses of FMT (1×10⁷ pfu) in the brain,we found no signs of cell death and comparable inflammatory responses tothose of saline injected control mice (FIG. 2E). This differeddramatically from VSV injected animals, which displayed a strikingincrease in inflammatory cells, condensed nuclei, and a perforatedmorphology. Due to its lack of neurotoxicity and potent CNS tumourkilling capacity, we elected to proceed with FMT as a platform todevelop as a novel oncolytic against GBM.

FIG. 2 shows the FMT Safety Profile A) Groups of 5 Balb/C mice wereinjected sterotaxically into the right striatum of the brain withincreasing doses of rhabdoviruses as indicated and monitored for signsof distress including weight loss, piloerection, hind leg paralysis,morbidity and mortality. Maximal Tolerable Dose (MTD) was determined tobe the highest dose not resulting in durable morbidity as measured bybehaviour and weight. LD₅₀ was determined using the Spearman Karbermethod. FMT is unique among rhabdoviruses as a non-neurotoxic virus. B)To detect any cognitive deficiencies in the striatum, motor function wasassessed by rotorod apparatus which measures latency to fall off anaccelerating rod. Motor function is indistinguishable from controlsafter intracerebral injection of FMT. C) Groups of 3-5 Balb/C mice wereinjected once intravenously in half log increments of virus ranging from3×10⁶ pfu to 3×10⁹ pfu. The animals were monitored for signs of distressincluding weight loss, morbidity, piloerection, hind-limb paralysis andrespiratory distress. FMT shows a very high MTD of 1×10⁹ pfu whendelivered systemically. D) Animals sacrificed 3 monthspost-intracerebral inoculation demonstrate no viable virus in the brainhomogenates. Limit of detection is 101. E) Balb/C mice were inoculatedintracerebrally with the indicated viruses (1×10⁷ pfu) and sacrificed 2days post treatment. Little to no inflammation is visible and no cellloss is detectable following FMT treatment. This is in contrast to VSVwhich shows significant neuronal loss (empty spaces, inset).

Example 3: Farmington Virus Potently and Selectivity Kills Brain TumourCells

Wild-type FMT isolates demonstrate attenuation in normal primary cellswhile maintaining potent glioma cell killing capacity (FIG. 3A). Toevaluate the clinical relevance of our novel oncolytic rhabdoviruses totreat brain cancer, we examined whether FMT could kill freshly derivedpatient tumour samples. Cell cultures isolated from 3 patients withprimary glioblastoma multiforme were infected with FMT and 48 h later,viability assays demonstrated that FMT virus was potently cytotoxic to 2of 3 patient tumour explants (FIG. 3A). To test the killing capacity ofthe FMT isolates we performed cell killing assays on normal humanastrocytes (NHA) and 3 GBM tumour cell lines (FIG. 3B). While wild typeMRB was very potent against all of the GBM cell lines, it was alsohighly lytic against both primary normal human astrocytes (NHA).Remarkably, FMT demonstrated the greatest therapeutic index, withpotency rivaling MRB in the majority of GBM lines while remaining highlyattenuated in NHA and GM38 primary cell lines (FIG. 3C). Thisdemonstrates that FMT virus is a potent and selective oncolytic viruswhen tested against brain cancer cell lines.

In FIG. 3 we show A) Summary of FMT in vitro cytotoxicity showing potentactivity against primary glioblastoma patient samples and establishedhuman and mouse brain tumour cell lines, while remaining attenuatedagainst normal cells. B) FMT is a potent and selective killer ofglioblastoma cell lines. Viability was assayed using Alamar blue assay72h post treatment. Error bars represent SEM of 4 biological replicates.C) Detailed assessment of FMT potency against tumour and normal cells.EC50 (moi=multiplicity of infection) represents the ratio of virus:cellrequired to kill 50% of cultured cells in a 72 time frame as measuredusing an Alamar Blue viability assay.

Example 4: Farmington Virus is Efficacious in Xenograft and SyngeneicModels of Glioblastoma

We next sought to determine the in vivo efficacy of our candidateviruses in mouse models of glioblastoma. After adapting human U87MGglioma cells for bioluminescent imaging, we established an intracerebralU87MG glioma model in athymic mice and we examined the IV and ICefficacy of FMT in this model (FIG. 4A-B). Specifically, animals weretreated with a single FMT dose IC (1×10⁵) or IV (5×10⁸ pfu) 14 days postimplantation. Three days after the first treatment we observed asignificant decrease in tumour burden with a greater decrease observedby day 7 (FIG. 4A). Interestingly the spinal metastases in this modelare completely cleared in all tumour bearing animals. In contrast,animals treated with UV inactivated virus had a significant increase intumour burden by day 7 at which point they started exhibitingneurological symptoms from their brain tumours (FIG. 4B). All IV treatedanimals responded to treatment with 4 of 11 durably cured and survivingbeyond 100 days post treatment. Most IC treated animals responded totreatment (10 of 16) with a significant (˜2 fold) increase in time todeath. Moreover we also used fluorescent microscopy to visualize tumourexplants of mock-infected animals and durably cured animals. While wedetect a strong GFP expressing glioma tumour in mock-infected animals,there is a clear absence of GFP signal in FMT treated animals (FIG. 4C).To complement our studies of viral efficacy in immunocompromisedanimals, we tested FMT in a mouse CT-2A syngeneic glioma model. Unlikexenograft models in which human gliomas grow expansively, CT-2A gliomasare infiltrative similar to what is observed clinically. We establishedthe CT-2A glioma model by stereotactically injecting 2×10⁵ cells intothe striatum (right frontal lobe) of C57BL/6 mice. Since treatmentstypically commence in human patients after they present with clinicalsymptoms of GBM, we sought to examine the effect of FMT at exactly thetime when animals exhibit outward symptoms. In the CT-2A animals beginto show symptoms 15-20 days post implantation. These symptoms includeincreased intracranial pressure, lethargy, motor function, piloerection,and hunched posture. Accordingly, 19 days post implantation C57BL/6animals were treated with FMT IV (5×10⁸ pfu thrice weekly for 2 weeks)or with a single IC dose of FMT (2×10⁷ pfu). Most animals responded toboth the treatment regimens, durable cures achieved in 3 of 11 IC and 2of 10 IV treated animals in this challenging model of advanced GBM (FIG.4D). Thus FMT virus demonstrates efficacy in preclinical models of braincancer.

FIG. 4 shows FMT In Vivo Efficacy in Preclinical Models of GlioblastomaA) Bioluminescence-adapted U87MG human gliomblatsoma cells (1e6) werestereotxically implanted into right striatum of CD-1 nude mice. After 2weeks animals were either treated with a single dose of FMTintravenously (IV—5×10⁸ pfu) or intracranially (IC—1×10⁵ pfu) andmonitored by IVIS bioluminescence imaging. Disseminated tumours in allmice treated with FMT regress rapidly within 3-7 days and becomeundetectable in the spinal cord. B) Kaplan Meir survival plot of animalstreated with a single dose IC (1×10⁵ pfu) resulting in a doubling ofmean time to death (Log rank test p=0.0001) or IV (5×10⁸ pfu) resultingin durable cures in 40% of animals (Log rank test p=0.0001). C)Fluorescence micrograph of a mock infected mouse brain with anorthotopic GFP tagged U87MG tumour (top 2 panels) versus a FMT treatedbrain (bottom 2 panels). GFP expressing tumour is clearly visible insagittal sections of untreated mice, while FMT treatment results in nodetectable GFP tumour signal, confirming tumour regression. D) Syngeneicmouse glioblastoma tumour model using the CT2A cell line. Here again,tumours were allowed to establish until the point where mice began todie from their tumour burden. Both IC (one dose 2×10⁷ pfu) and IV (6doses 5×10⁸) treatment doubled mean time to death and resulted in >20%durable cures.

Example 5: Farmington Virus Induces Anti-Tumour Immunity

Emerging evidence is demonstrating that the oncolytic effect of manyviruses, including oncolytic rhabdoviruses, is due in part to theinduction of anti-tumor immunity. To explore the possibility that FMTvirus induces multiple mechanisms for tumor destruction in vivo, weasked whether treating immunocompetent tumor-bearing mice with FMT virusevokes anti-tumor immunity. To begin, we performed a “re-challenge”experiment, where C57/BL6 mice previously harboring CT-2A tumors thathad been successfully treated with IC FMT virus infusions were injectedfor a second time with CT-2A cells directly into the brain. In theseexperiments, previously cured mice uniformly rejected the cells (FIG.5A), demonstrating that they had acquired long-lasting immunity towardsCT-2A antigen(s). Next, we examined the role of cytotoxic T-lymphocytes(CTLs) in the anti-tumor response elicited by FMT virus. Mice wereinoculated with CT-2A cells in their brain, and then CTLs were removedusing antibodies directed towards CD8 concomitant with FMT virustreatment. Consistent with recent data from other labs using differentoncolytic agents, these experiments showed that FMT virus inducedcomplete responses only when CTLs were present (FIG. 5B). Thus, inaddition to directly lysing MG cells, these data demonstrate that FMTvirus induces a potent and long-lasting CTL-mediated anti-MG immuneresponse in immunocompetent mice.

In FIG. 5A, C57/B6 mice were implanted with CT2A murine glioma cellsinto the striatum (3×10⁵ cells). Mice were treated with a single dose ofFMT (2×10⁷ pfu) and subsequently cured of their initial tumour. After 6months, these mice were challenged with CT2A cells implanted into thestriatum. Naive mice were implanted with CT2A cells as a control.Bioluminescent imaging to monitor CT2A tumour growth showed that micethat had previously been cured of the tumours completely rejectedsubsequent CT2A tumour growth, while naive mice grew tumours with theexpected kinetics. In FIG. 5B) 057/B6 mice were implanted with CT2Amurine glioma cells into the striatum (3×10⁵ cells) and allowed to growtumours for 14 days. One group mice received anti-CD8 polyclonal seruminjections to remove CD8+ T cells or matched pre-immune serum as acontrol. Both groups were treated with a single intracranial dose of FMT(2×10⁷ pfu) to induce tumour regressions. All mice responded with tumourregression as measured using bioluminescent tumour imaging (not shown),but mice that had been stripped of their CD8+ T cells all eventuallyregrew tumours and failed therapy.

Example 6: Farmington Virus Productively Infects Tumor and Normal Cells

Without exception, the therapeutic index associated with existingoncolytic viruses is due to differential infection and/or productivityin tumor versus normal cells. In the case of rhabdoviruses, currentoncolytic strains can “sense” cancer-specific defects in type I IFNsignalling, which renders them selectively productive in transformedcells. Unfortunately, current oncolytic rhabdoviruses are highlyneurotoxic when delivered local-regionally into the CNS. Given that FMTvirus is safe when infused IC and is genetically very distinct from theexisting agents, we suspected that its mechanism for tumor-specificdestruction must be something other than heightened productivitysecondary to IFN defects in tumor cells. To begin to evaluate thishypothesis, we infected SNB19 and NHA cells with rec-FMT-GFP virus andevaluated GFP expression and viral protein production over time.Fluorescence microscopy and immunoblot analyses clearly demonstratedthat FMT virus does not distinguish tumor from normal via differentialinfectivity or viral protein production (FIG. 6A-B). Specifically, bothGFP and FMT protein expression became easily detectable 6.5-16 hoursafter infection and continued to be strongly expressed 72 hours later,irrespective of the transformed state of the infected cell line. Inparallel, we performed one-step growth evaluation of FMT virus invarious tumor and normal cell lines to examine productivity. Theseexperiments showed that infectious FMT virus particles are producedquickly (within 6.5 hours) and to high titers (˜10⁸ pfu) in both tumorand normal cell lines (Figure. 6B). Collectively, these date demonstratethat, in stark contrast to the existing VSV Δ51 and Maraba MG1 series ofoncolytic rhabdoviruses, FMT virus is equally productive in normal ascompared to tumor cells.

We thus interrogated the interaction between FMT virus and the type Iinterferon response. GFP-expressing rec-FMT or Maraba-Δ51 virus wasadded at low moi to a GM38 cell monolayer, and plaque and infectiousfoci size were measured. As expected, Maraba-Δ51 infection did notinfect or kill GM38 cells unless the innate immune response was blockeda priori by treatment with the type I interferon inhibitor B18R proteinderived from vaccinnia virus (VV-B18R; FIG. 6C-D). In contrast, FMTvirus formed moderately-sized infectious foci in the GM38 cells, but didnot kill them, and was unaffected by B18R pre-treatment. Furthermore,unlike Maraba-Δ51, FMT virus completely blocked type I interferonproduction in PC3 cells as measured in an interferon bioassay (FIG. 6E).Collectively, these results indicate that, in contrast to the currentarsenal of genetically engineered oncolytic rhabdoviruses, FMT viruspotently blocks the human type I interferon response and canproductively infect both normal and tumor cell lines, which pointstowards a novel cancer-selective mechanism for this oncolytic agent.

In FIG. 6 is shown A) Western blot demonstrating similar kinetics of FMTvirus infection and protein production in both human brain tumour cells(SNB19) and primary normal human astrocytes (NHA). B) Single step growthcurve showing identical virus replication and virion productivity fromtumour (SNB19) and normal (NHA) cells following infection with FMT. C)Fluorescence Microscopy of GFP-expressing rec-FMT or Maraba-Δ51 virusadded to a GM38 cell monolayer, and plaque and infectious foci size weremeasured showing that Maraba-Δ51 infection did not infect or kill GM38cells unless the innate immune response was blocked by treatment withthe type I interferon inhibitor B18R protein derived from vaccinniavirus (VV-B18R) while FMT virus formed infectious foci in GM38 cells andwas unaffected by B18R pre-treatment. D) Size of Infectious focidetermined from fluorescence microscopy. E) Interferon bioassay in PC3cells shows that unlike Maraba-Δ51, FMT blocks type I interferonproduction.

Example 7: Farmington Virus Selectively Induces Apoptosis in Tumor Cells

We used the NT2 cell system that consists of transformed NT2teratocarcinoma cells which can be induced to differentiate into postmitotic neurons with retanoic acid. Following infection with wild typeVSV or FMT we observed that these viruses infected and producedinfectious progeny to the same degree in either cancerous ornon-cancerous forms of the NT2 cells (FIG. 7A). However, although theseviruses were potently cytotoxic to malignant NT2 cells, FMT showedalmost no cytotoxicity in differentiated post mitotic NT2 cells (FIG.7B). This indicated that in contrast to other oncolytic rhabdoviruseslike VSV, FMT virus appeared to have a unique mechanism of tumourselectivity functioning at the level of cytotoxicity.

Rhabdoviruses kill permissive cells by apoptosis, activated throughvirus-mediated degradation of key BH3-only proteins, an event whichultimately engages the apical intracellular and extracellular caspasesthat initiate the irreversible cell death cascade. To ascertain whetherFMT virus selectively kills tumor cells via differential induction ofapoptosis, we evaluated the activation status of key proteins in theapoptotic heirarchy. As expected, the surrogate apoptosis marker PARP aswell as the downstream effector caspase 3 were strongly activated inSNB19 but not NHA cells treated with FMT virus (FIG. 7C), indicating thepresence of apoptosis. Moreover, the activator caspases 8 and 9 wereengaged in the tumor cells, but not in the normal cells. The NT2 cellsystem data is consistent with this apoptosis data in the transformedversus normal cell lines showing that cell death induced by FMT virus isdependent not on the productivity of the virus infection within, butrather the anti-apoptotic threshold of, the infected cell.

In FIG. 7A, both pre and post differentiated cell types were permissiveto infection by wt VSV and wt FMT virus, producing infectious particlesin the 10⁶-10⁸ range. B) shows that only VSV was cytolytic against theneuron. C) Western blot of several components of the cellular apoptoticsignaling cascade following infection of either tumour (SNB19) or normalcells (NHA). FMT appears to initiate the activation (cleavage) ofCaspase 8, Caspase 9, BID, and PARP only in tumour cells. To ourknowledge, this is the first report of an oncolytic virus whose activityis restricted not at the level of infectivity, but at the level ofselective initiation of cell death. D) Schematic of cellular apoptosissignaling cascade. Proteins that are cleaved during activation aredepicted in orange and correspond to those included in our westernblotting array in panel C.

In the preceding description, for purposes of explanation, numerousdetails are set forth in order to provide a thorough understanding ofthe examples. However, it will be apparent to one skilled in the artthat these specific details are not required. Equivalent changes,modifications and variations of some examples, materials, compositionsand methods can be made within the scope of the present technology, withsubstantially similar results.

The above-described examples are intended to be exemplary only.Alterations, modifications and variations can be effected to theparticular embodiments by those of skill in the art without departingfrom the scope, which is defined solely by the claims appended hereto.

1. A method for inducing an immunogenic response in a patient, themethod comprising administering to the patient: A) an isolated viralparticle having a genome comprising open reading frames that encode: aprotein having a sequence that is at least 95% identical to SEQ ID NO:3; a protein having a sequence that is at least 95% identical to SEQ IDNO: 4; a protein having a sequence that is at least 95% identical to SEQID NO: 5; a protein having a sequence that is at least 95% identical toSEQ ID NO: 6; and a protein having a sequence that is at least 95%identical to SEQ ID NO: 7, wherein at least one of the encoded proteinscontains at least one non-naturally occurring substitution modificationrelative to SEQ ID NO: 3, 4, 5, 6, or 7; B) an isolated viral particlethat produces a cDNA polynucleotide when the viral particle is in a hostcell, the cDNA polynucleotide comprising: a sequence that is at least95% identical to SEQ ID NO: 8; a sequence that is at least 95% identicalto SEQ ID NO: 9; a sequence that is at least 95% identical to SEQ ID NO:10; a sequence that is at least 95% identical to SEQ ID NO: 11; and asequence that is at least 95% identical to SEQ ID NO: 12, wherein atleast one of the sequences contains at least one non-naturally occurringsubstitution modification relative to SEQ ID NO: 8, 9, 10, 11, or 12; C)an isolated viral particle that produces a cDNA polynucleotide when theviral particle is in a host cell, wherein the cDNA polynucleotidecomprises: a sequence that is at least 95% identical and is less than99% identical to SEQ ID NO: 1 and contains at least one non-naturallyoccurring substitution modification relative to SEQ ID NO: 1; or D) anisolated viral particle comprising an RNA polynucleotide comprising asequence that is at least 95% identical and is less than 99% identicalto SEQ ID NO:2 and contains at least one non-naturally occurringsubstitution modification relative to SEQ ID NO:
 2. 2. The methodaccording to claim 1, wherein the method comprises administering to thepatient the isolated viral particle having a genome comprising openreading frames that encode: a protein having a sequence that is at least95% identical to SEQ ID NO: 3; a protein having a sequence that is atleast 95% identical to SEQ ID NO: 4; a protein having a sequence that isat least 95% identical to SEQ ID NO: 5; a protein having a sequence thatis at least 95% identical to SEQ ID NO: 6; and a protein having asequence that is at least 95% identical to SEQ ID NO: 7, wherein atleast one of the encoded proteins contains at least one non-naturallyoccurring substitution modification relative to SEQ ID NO: 3, 4, 5, 6,or
 7. 3. The method according to claim 2, wherein the open readingframes encode: a protein having a sequence that is at least 99%identical to SEQ ID NO: 3; a protein having a sequence that is at least99% identical to SEQ ID NO: 4; a protein having a sequence that is atleast 99% identical to SEQ ID NO: 5; a protein having a sequence that isat least 99% identical to SEQ ID NO: 6; and a protein having a sequencethat is at least 99% identical to SEQ ID NO:
 7. 4. The method accordingto claim 1, wherein the method comprises administering to the patientthe isolated viral particle that produces a cDNA polynucleotide when theviral particle is in a host cell, the cDNA polynucleotide comprising: asequence that is at least 95% identical to SEQ ID NO: 8; a sequence thatis at least 95% identical to SEQ ID NO: 9; a sequence that is at least95% identical to SEQ ID NO: 10; a sequence that is at least 95%identical to SEQ ID NO: 11; and a sequence that is at least 95%identical to SEQ ID NO: 12, wherein at least one of the sequencescontains at least one non-naturally occurring substitution modificationrelative to SEQ ID NO: 8, 9, 10, 11, or
 12. 5. The method according toclaim 4, wherein the cDNA polynucleotide further comprises at least onepromoter sequence.
 6. The method according to claim 1, wherein themethod comprises administering to the patient the isolated viralparticle that produces a cDNA polynucleotide when the viral particle isin a host cell, wherein the cDNA polynucleotide comprises: a sequencethat is at least 95% identical and is less than 99% identical to SEQ IDNO: 1 and contains at least one non-naturally occurring substitutionmodification relative to SEQ ID NO:
 1. 7. The method according to claim1, wherein the method comprises administering to the patient theisolated viral particle comprising an RNA polynucleotide comprising asequence that is at least 95% identical and is less than 99% identicalto SEQ ID NO:2 and contains at least one non-naturally occurringsubstitution modification relative to SEQ ID NO:
 2. 8. The methodaccording to claim 1, wherein the isolated viral particle isadministered to the patient intrathecally, intravenously or viaintracranial injection.
 9. The method according to claim 1, wherein theisolated viral particle is administered to the patient inside theblood/brain barrier by intracranial injection.
 10. A method for inducingan immunogenic response in a patient, the method comprisingadministering to the patient: A) an isolated viral particle having agenome comprising open reading frames that encode: a protein having asequence that is at least 95% identical to SEQ ID NO: 3; a proteinhaving a sequence that is at least 95% identical to SEQ ID NO: 4; aprotein having a sequence that is at least 95% identical to SEQ ID NO:5; a protein having a sequence that is at least 95% identical to SEQ IDNO: 6; a protein having a sequence that is at least 95% identical to SEQID NO: 7, and at least one heterologous protein; B) an isolated viralparticle that produces a cDNA polynucleotide when the viral particle isin a host cell, the cDNA polynucleotide comprising: a sequence that isat least 95% identical to SEQ ID NO: 8; a sequence that is at least 95%identical to SEQ ID NO: 9; a sequence that is at least 95% identical toSEQ ID NO: 10; a sequence that is at least 95% identical to SEQ ID NO:11; a sequence that is at least 95% identical to SEQ ID NO: 12; and asequence that encodes at least one heterologous protein; C) an isolatedviral particle that produces a cDNA polynucleotide when the viralparticle is in a host cell, wherein the cDNA polynucleotide comprises:(i) a sequence that is at least 95% identical to SEQ ID NO: 1 and (ii) asequence that encodes at least one heterologous protein; or D) anisolated viral particle comprising an RNA polynucleotide comprising: (i)a sequence that is at least 95% identical to SEQ ID NO:2 and (ii) asequence that encodes at least one heterologous protein.
 11. The methodaccording to claim 10, wherein the method comprises administering to thepatient the isolated viral particle having a genome comprising openreading frames that encode: a protein having a sequence that is at least95% identical to SEQ ID NO: 3; a protein having a sequence that is atleast 95% identical to SEQ ID NO: 4; a protein having a sequence that isat least 95% identical to SEQ ID NO: 5; a protein having a sequence thatis at least 95% identical to SEQ ID NO: 6; a protein having a sequencethat is at least 95% identical to SEQ ID NO: 7, and at least oneheterologous protein.
 12. The method according to claim 11, wherein theheterologous protein is an immunogenic protein.
 13. The method accordingto claim 11, wherein the open reading frames encode: a protein having asequence that is at least 99% identical to SEQ ID NO: 3; a proteinhaving a sequence that is at least 99% identical to SEQ ID NO: 4; aprotein having a sequence that is at least 99% identical to SEQ ID NO:5; a protein having a sequence that is at least 99% identical to SEQ IDNO: 6; a protein having a sequence that is at least 99% identical to SEQID NO: 7, and at least one heterologous protein.
 14. The methodaccording to claim 10, wherein the method comprises administering to thepatient the isolated viral particle that produces a cDNA polynucleotidewhen the viral particle is in a host cell, the cDNA polynucleotidecomprising: a sequence that is at least 95% identical to SEQ ID NO: 8; asequence that is at least 95% identical to SEQ ID NO: 9; a sequence thatis at least 95% identical to SEQ ID NO: 10; a sequence that is at least95% identical to SEQ ID NO: 11; a sequence that is at least 95%identical to SEQ ID NO: 12, and a sequence that encodes at least oneheterologous protein.
 15. The method according to claim 14, wherein thecDNA polynucleotide further comprises at least one promoter sequence.16. The method according to claim 14, wherein the heterologous proteinis an immunogenic protein.
 17. The method according to claim 10, whereinthe method comprises administering to the patient the isolated viralparticle that produces a cDNA polynucleotide when the viral particle isin a host cell, wherein the cDNA polynucleotide comprises: (i) asequence that is at least 95% identical to SEQ ID NO: 1 and (ii) asequence that encodes at least one heterologous protein.
 18. The methodaccording to claim 17, wherein the heterologous protein is animmunogenic protein.
 19. The method according to claim 11, wherein themethod comprises administering to the patient the isolated viralparticle comprising an RNA polynucleotide comprising: (i) a sequencethat is at least 95% identical to SEQ ID NO: 2 and (ii) a sequence thatencodes at least one heterologous protein.
 20. The method according toclaim 19, wherein the heterologous protein is an immunogenic protein.